藤黄酸通过调节PPARγ表达与内质网应激抑制弥漫性大B细胞淋巴瘤生长

Gambogic acid inhibits the growth of diffuse large B-cell lymphoma by regulating PPARγ expression and endoplasmic reticulum stress

目的:探讨藤黄酸(gambogic acid,GA)对弥漫性大B细胞淋巴瘤(diffuselarge B-cell lymphoma,DLBCL)细胞增殖活性与凋亡的影响,以及对小鼠DLBCL肿瘤生长的影响与其机制研究。方法:将培养的DLBCL细胞系OCI-ly8和SUDHL-4随机分成空白对照组与0.5、1.0、2.5、5.0、10.0μmol/L藤黄酸组,CCK-8实验和Annexin V-FITC/PI实验分别检测各处理组细胞活性和凋亡情况。通过小鼠皮下注射SUDHL-4细胞悬液构建DLBCL模型,将造模成功的10只小鼠随机分为对照组与藤黄酸组,每组5只,藤黄酸组小鼠尾静脉注射藤黄酸(1 mg/kg),对照组小鼠注射等量生理盐水,每日注射一次,持续14天。期间每隔一日测量小鼠的体重与肿瘤体积,14天后取小鼠肿瘤组织并称重,免疫组织化学染色检测肿瘤组织中过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)表达,Western blot检测内质网应激标记蛋白GRP78、CHOP、Caspase-4、p-JNK、p-PERK的表达水平,TUNEL法检测细胞凋亡情况。结果:经不同浓度藤黄酸干预的OCI-ly8和SUDHL-4细胞存活率较空白对照组均显著下降(P<0.05),细胞凋亡率显著增加(P<0.05),具有剂量依赖性。造模成功后经藤黄酸干预的小鼠体重较对照组小鼠体重差异无统计学意义(P>0.05),而肿瘤体积显著减小(P<0.05);14天后小鼠肿瘤组织重量显著低于对照组(P<0.05);Ki-67阳性细胞表达率较对照组显著下降(P<0.05),PPARγ阳性细胞表达率较对照组显著升高(P<0.05);肿瘤组织中GRP78、CHOP、Caspase-4、p-JNK、p-PERK蛋白表达水平较对照组均显著增加(P<0.05);凋亡细胞数较对照组凋亡细胞数显著增加(P<0.05)。结论:藤黄酸可抑制DLBCL细胞的增殖活性、促进细胞凋亡并抑制小鼠DLBCL的生长,其机制可能与调控PPARγ表达与内质网应激相关。

Objective:To investigate the effects of gambogic acid on the proliferation and apoptosis of diffuse large B-cell lymphoma(DLBCL)cells,as well as the effects and mechanism of tumor growth in DLBCL mice.Methods:The cultured DLBCL cell lines OCI-ly8 and SUDHL-4 were randomly divided into a blank control group and 0.5,1.0,2.5,5.0,10.0μmol/L gambogic acid groups.The CCK-8 test and Annexin V-FITC/PI test were used to detect the cell activity and apoptosis in each treatment group.The DLBCL model was constructed by subcutaneously injecting SUDHL-4 cell suspension in mice,and 10 mice successfully modeled were randomly divided into a control group and a gambogic acid group,with 5 mice in each group.Gambogic acid group mice were injected with gambogic acid(1 mg/kg)in the tail vein.The mice in the control group were injected with the same amount of normal saline,injection once a day for 14 days.During the period,the body weight and tumor volume of the mice were measured every other day.After 14 days,the tumor tissues of the mice were taken and weighed.Immunohistochemical staining was used to detect the expression of peroxisome proliferator-activated receptorγ(PPARγ)in tumor tissues.Western blot was used to detect the expression levels of endoplasmic reticulum stress marker proteins GRP78,CHOP,Caspase-4,p-JNK,p-PERK.TUNEL method was used to detect apoptosis.Results:The survival rates of OCI-ly8 and SUDHL-4 cells with different concentrations of gambogic acid were significantly lower than that of the blank control group(P<0.05),and the apoptosis rate was significantly increased(P<0.05),which was dose-dependent.After successful modeling,the body weight of mice treated with gambogic acid was not significantly different from that of the control group(P>0.05),while the tumor volume was significantly reduced(P<0.05).After 14 days,the weight of tumor tissue in mice was significantly lower than that in the control group(P<0.05).The expression rate of Ki-67 positive cells was significantly lower than that of the control group(P<0.05),and the expression rate of PPARγpositive cells was significantly higher than that of the control group(P<0.05).The expression levels of GRP78,CHOP,Caspase-4,p-JNK and p-PERK protein in tumor tissue were significantly increased compared with the control group(P<0.05).The number of apoptotic cells increased significantly compared with the number of apoptotic cells in the control group(P<0.05).Conclusion:Gambogic acid can inhibit the proliferation of DLBCL cells,promote apoptosis and inhibit tumor growth in DLBCL mice.The mechanism may be related to the regulation of PPARγexpression and endoplasmic reticulum stress.